Single-Domain Antibody Strengthening Fusion Protein Vh-Ldp-Ae

ABSTRACT

The present invention relates to a novel antibody-based targeting drug—a single domain antibody energized fusion protein VH-LDP-AE with potent cancer cell killing activity, anti-angiogenic activity, and anti-cancer therapeutic efficacy, to a method for producing the same, and use thereof in manufacture of an anti-tumor medicament.

FIELD OF THE INVENTION

The present invention relates to a novel antibody-based, targeted drugwith potent cancer cell killing activity, anti-angiogenic effects andantitumor efficacy—a single domain antibody energized fusion proteinVH-LDP-AE, to a method for producing the same, and use thereof inmanufacture of an anti-tumor medicament.

BACKGROUND OF THE INVENTION

Type IV collagenases can degrade the extracellular matrix componentssuch as type IV collagen, destroy the integrality of basement membraneand extracellular matrix and play an important role in tumor growth,invasion and metastasis. There is a high expression in various kinds oftumor tissue and cells such as human prostate cancer, colorectal cancer,breast cancer, melanoma, pancreatic cancer, etc., and the inhibition oftype IV collagenase activity can suppress the growth and metastasis oftumor. The present invention relates to a single domain antibody whichderived from the monoclonal antibody 3G11 directed against type IVcollagenase, which is immunoreactivitily positive to various kinds oftumor cells and has the capability of binding specifically to variouskinds of human tumor tissues.

Single domain antibodies (dAbs) are the smallest functional bindingunits of an antibody, which corresponds to the variable regions of theheavy (VH) or the light (VL) chains of human antibodies. As the newgeneration of therapeutic antibodies after intact antibodies, Fabfragments, and single chain antibodies, single domain antibodies have amolecular weight of 11˜13 kDa, or approximately one-twelfth the size ofa full antibody (150 kDa). Compared with intact antibodies, singledomain antibodies have much smaller molecular size and this propertydetermined that they not only can better penetrate into theintercellular space of solid tumor, but also own more uniformbio-distribution and lower immunogenicity in tumor. However, due to thelack of Fc portion and hence loss of the capability of trigger effectorfunctions, dAbs need to be used in combination with the “warhead” drugin cancer therapy. DAbs are promising as an excellent vehicle candidatefor targeted therapy of cancer.

As the highly potent “warhead” drug, lidamycin (LDM, also named C-1027or C1027) is an enediyne-containing antibiotic produced by Streptomycesglobisporus (accession number: CGMCC No. 0135) which was isolated from asoil sample collected in Qianjiang County, Hubei Province, China. LDM isone of the macromolecular peptide antibiotics which displays the mostpotent cytotoxicity against tumor cells reported hitherto. In vivoexperiments had demonstrated that LDM remarkably inhibited the growth ofcolon carcinoma 26 in mice, and also showed potent inhibitory effect onthe growth of human cancer xenografts such as hepatoma Bel-7402 andcecum carcinoma Hce-8693 in nude mice (Chinese Journal of Antibiotics1994, 19 (2):164-168). The LDM molecule consists of two moieties, one isenediyne chromophore (AE, active enediyne) which is labile andresponsible for cytotoxicity; another is apoprotein (LDP) containing 110amino acid residues, which plays a pivotal role in keeping the stabilityof chromophore. There is a non-covalent binding between chromophore andapoprotein. Although the binding is specific and fast, the chromomhoreand apoprotein can be separated and molecular reconstituted. Due to theunique molecular structure, LDM is a perfect “warhead” candidate forconstructing novel antibody-based, targeting drugs (Acta AcademiaeMedicinae Sinicae, 2001, 23 (6): 563-567).

The targeted cancer therapy has better efficacy because it can rightdeliver a drug to cancer tissue and decrease the toxicity to normalcells. Monoclonal antibodies have been used as carriers for drug intargeted cancer therapy. However, clinic studies demonstrated that thelarge mAb molecules were unable to penetrate tumor effectively to reachthe region deep within the solid tumor; as a result, the solid tumor isrelatively resistant to targeted immunotherapy of cancer. In addition,the high normal-tissue/tumor distribution ratio of large antibodymolecules and high immunogenecity of mouse antibody both limited theirclinical use. If minimize the antibody size while preserve its antigenaffinity, and at the same time, load it with “warhead” drug which haspotent cytotoxic activity against tumor cells, the “down-sized” antibodymolecule carrying the “warhead” drug will be able to reach efficientlythe tumor target and the region deep within the solid tumor. Thisstrategy may not only get over the resistance problem of solid tumoragainst antibody-based therapy and reduce the immunogencity, but alsodecrease the effective concentration of the “warhead” drug and make itwork at much lower concentrations. So the therapeutic effects will beimproved. According to the strategy, using the advantage of singledomain antibody and the characteristic of LDM which can be separated andreconstituted, the present inventors manufactured a novel, down-sized,and highly effective dAb-based energized fusion protein VH-LDP-AE bygene recombination and molecular reconstitution as a novelantibody-targeted drug which demonstrates potent antitumor activity.

DETAILED DESCRIPTION OF THE INVENTION

The conjugates of large monoclonal antibody and drug are hard to reachthe tumor cells deep within the solid tumor through the endothelium ofcapillary vessels and extracellular space in solid tumor in tumortherapy. It is valuable to develop a down-sized and highly-effectiveantibody drug for improving the therapeutic efficacy (Yong-su Zhen.Advances in Research on Monoclonal Antibody Agents for Cancer Therapy.Acta Academiae Medicinae Sinicae, 2000, 22 (1): 9-13). Using VH domainof mAb 3G11 which directed against type IV collagenase as a carrier andthe highly-potent antitumor antibiotic LDM as the “warhead”, the presentinventors, by DNA recombination and molecular reconstitution,manufactured the novel antibody-targeted, dAb-based, and energizedfusion protein VH-LDP-AE characterized as down-sized molecule and highefficacy. VH-LDP-AE can bind specifically to tumor cells, inhibitangiogenesis, and also demonstrate good antitumor efficacy in animalexperiments.

In one respect, the present invention relates to the saiddomain-antibody-based and energized fusion protein VH-LDP-AE, whichcomposed of fusion protein VH-LDP and the active enediyne (AE)chromophore of lidamycin, wherein the said VH-LDP protein consists of VHdomain of mAb 3G11 directly against type IV collagenase, a flexibleprotein spacer (GGGGS), an apoprotein LDP of LDM, and six-histidine tag(His₆-Tag) in the carboxyl terminal.

1. Single-Domain Antibody Fusion Protein VH-LDP

Specifically, the gene encoding the fusion protein VH-LDP is 732 bp (SEQID NO: 1) and it encodes 243 amino acids (SEQ ID NO: 2). The molecularweight of VH-LDP is 25.4 kDa.

In the present invention, the VH moiety in fusion protein VH-LDP derivedfrom the heavy chain variable region of monoclonal antibody 3G11(hybridoma strain of 3G11 has been deposited in China GeneralMicrobiological Culture Collection Center with accession number CGMCCNo. 0831). Previous experiments demonstrated that the immunoreactivityof mAb 3G11 was positive to various human tumor tissues and showedtargeted distribution in human lung cancer xenograft transplanted intonude mice (Yao Dai, Bing Jia, Yong-su Zhen, et al. Immunoscintigraphy ofanti-type IV collagenase monoclonal antibody in nude mice bearing humanlung cancer xenograft, Chinese Journal of Cancer, 2003, 22(12):1243-1248). The present inventors found that single domain antibodyfusion protein VH-LDP owned part of antigen-binding and -inhibitingactivity of the intact antibody. It can bind specifically to cancercells and inhibit the activity of type IV collagenase.

2. The Active Enediyne Chromophore AE

The molecular weight of LDM is 11349.1120 Dalton, which is consisted ofa LDP with 10505.7830 Dalton and chromophore AE with 843.3295 Dalton.

Chemical Name of Chromophore AE:

-   (2R,7S,9R,10R)-7-Amino-7,8-(2*-chloro-6*-hydroxy-1*,4*-phenylene)-10-(4′-deoxy-4′-dimethylamino-5′,5′-dimethyl-ribopyranosido)-4,8-dioxa-5-oxo-1,11,13-trien-15,18-diyn-tricyclo-   [7,7,3,0^(10,14)]-2-nondecanyl-2″,3″-dihydro-7″-methoxy-2″-methylene-3″-oxo-1″,4″-benzo    xazine-5″-carboxylate

Molecular formula of lidamycin: C₄₃H₄₂O₁₃N₃Cl

The chemical structure of active and aromatized lidamycin chromophore:

Two parts of lidamycin, LDP and chromophore, connecting with each otherspecifically and firmly through non-covalent binding, can be dissociatedand reconstituted to rebuild an energized molecule. The uniqueproperties of molecular constitution, low molecular weight of AE, andpotent bioactivity make lidamycin a promising “warhead” agent inconstructing new monoclonal antibody targeted drugs (Acta Acad Med Sin,2001, 23<6>: 563-567).

In one embodiment of the present invention, the molar ratio of saidfusion protein VH-LDP and active chromophore AE of LDM is 1:1 in thesaid dAb-based and energized fusion molecule VH-LDP-AE.

Another aspect of the present invention relates to the preparation ofenergized fusion protein VH-LDP-AE. In details, the fusion proteinVH-LDP was produced at first by DNA recombination in E. coli expressionsystem. The inventors found that the resultant fusion protein VH-LDP hadthe antigen-binding and antigen-inhibiting activity and can bindselectively to tumor tissue. And then, the domain-antibody-based andenergized fusion protein VH-LDP-AE was obtained through assemblingactive domain-antibody-based fusion protein VH-LDP with activechromophore AE of lidamycin that is obtained by the method of coldmethanol extraction. The molecular ratio of AE and VH-LDP is 5:1 and thevolume ratio of them is 1:50. The inventors surprisingly found thatVH-LDP-AE can potently kill the tumor cells and inhibit angiogenesis. Atthe same time, compared with the existing scFv-based energized fusionprotein, VH-LDP-AE would have better penetration into solid tumors,lower immunogenicity, stronger antitumor effects, or less risk ofinducing side-effects in clinical application due to its much smallersize.

On another respect, the present invention relates to use of the saidenergized fusion protein VH-LDP-AE in manufacture of a medicament foranti-angiogenesis and antitumor treatment.

On another respect, the present invention relates to a pharmaceuticalcomposition comprising therapeutically effective amount of energizedfusion protein VH-LDP-AE, and optionally, said pharmaceuticalcomposition further comprises pharmaceutical acceptable carrier andexcipient compatible to the administration route and dosage thereof.

On another respect, the present invention relates to a method fortreating malignant cancers, including the administration oftherapeutically effective amount of said energized fusion protein or thepharmaceutical composition of the present invention to the patient withtumor.

Some earlier studies showed that single VH domain can retain part ofantigen affinity, but isolated VH domain tended to congregate intosedimentation. The research in the present lab demonstrated that LDMitself is water soluble, and apoprotein of lidamycin alone could beexpressed solubly in E. Coli. The inventors attempted to make fusiongene in the form of VH-LDP by fusing VH domain against type IVcollagenase to apoprotein LDP of LDM, and then expressing the fusionprotein in E. coli. As a result, the present inventors surprisinglyfound that the fusion protein after refolding was soluble in PBSsolution, and most of them (75%) existed in monomer state. The resultshowed that the constructing method in the present invention overcameeffectively the adverse influence of VH congregation in solution.

As mentioned above, single-domain antibodies are the minimal functionalbinding units of antibody and are regarded as a new generation oftherapeutic antibodies. In the present invention, using type IVcollagenase as the target, single domain antibody derived from mAb 3G11as the carrier, potent antitumor antibiotic LDM as the “warhead”, theenergized fusion protein VH-LDP-AE was prepared by DNA recombination andmolecular reconstitution. The research results of this invention showedthat the energized fusion protein VH-LDP-AE, which consisted of VHdomain of mAb 3G11 against type IV collagenase and the antitumorantibiotic LDM not only retained the binding activity of intact mAb 3G11to type IV collagenase, killed tumor cells potently, and inhibitedangiogenesis, but also showed antitumor effects in animal experiments.Through searches, we found that VH-LDP-AE, with a molecular mass of 26.2kDa, is the minimal domain-antibody-based fusion protein with remarkabletherapeutic effects in animal experiments ever reported. VH-LDP-AEreaches a new level in molecule down-sizing of antibody-based drugs andis promising in clinical application.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Restriction enzymatic analysis of recombinant plasmidpET-VH-LDP.

Lane 1, DNA MW markers (DL15,000); lane 2, pET-30a(+); lane 3,recombinant plasmid pET-VH-LDP; 4, pET-30a(+)/NdeI+XhoI; 5,pET-VH-LDP/NdeI+XhoI; 6, pET-VH-LDP/NdeI+BamHI; 7,pET-VH-LDP/BamHI+XhoI; 8, DNA MW markers (DL2,000).

FIG. 2. SDS-PAGE (left) and Western-blot (right) analysis of eachfraction of fusion protein 1H-LDP.

Lane 1, low molecular weight marker; lane 2, total proteins of E. coliBL21star™ carrying plasmid pET-30a(+) after IPTG induction; lane 3,total proteins of recombinant E. coli CAMS/HLDFP before IPTG induction;lane 4, total proteins of recombinant E. coli CAMS/HLDFP after IPTGinduction; lane 5, medium sample of recombinant E. coli CAMS/HLDFP afterIPTG induction; lane 6, periplasmic fraction of recombinant E. coliCAMSIHLDFP after IPTG induction; lane 7, cytoplasmic soluble fraction ofrecombinant E. coli CAMS/HLDFP after IPTG induction; lane 8, cytoplasmicinsoluble fraction of recombinant E. coli CAMS/HLDFP after IPTGinduction.

FIG. 3. SDS-PAGE analysis of purification of fusion protein VH-LDP byImmobilized Metal Affinity Chromatography (IMAC).

Lane 1, low molecular weight marker; lane 2, total cell protein; 3,sample before applied to Ni²⁺ column; 4, non-desired proteins not boundto the resin; 5,6, fraction of non-desired proteins washed out by 1×Binding Buffer; 7, fraction of non-desired proteins washed out by 1×washing buffer; 8,9, fusion protein VH-LDP obtained by eluting with 1×Strip Buffer.

FIG. 4. Immunoreactivity of fusion protein VH-LDP with type IVcollagenase. ∘, fusion protein VH-LDP; ▴, scFv protein against type IVcollagenase.

FIG. 5. Immunoreactivity of fusion protein VH-LDP with human hepatomaSMMC-7721 cells. ∘, fusion protein VH-LDP; ▴, scFv protein against typeIV collagenase.

FIG. 6. Immunoreactivity of fusion protein VH-LDP with human oralepidermoid carcinoma KB cells. ∘, fusion protein VH-LDP; ▴, scFv proteindirectly against type IV collagenase.

FIG. 7. Immunoreactivity of fusion protein VH-LDP with solid tumor ofmouse hepatoma 22. A, negative control, with PBS instead of fusionprotein VH-LDP as the primary antibody; B Immunol histochemical stain ofnormal liver section of mice reacted with fusion protein VH-LDP; C,Immunol histochemical stain of hepatoma H 22 section reacted with fusionprotein VH-LDP; D, positive control, Immunol histochemical stain ofhepatoma H22 section reacted with scFv protein directly against type IVcollagenase.

FIG. 8, Gelatin zymography analysis of fusion protein VH-LDP in HT-1080cells. Lane 1, control, treated with PBS; lane 2, treated with 25 μM offusion protein VH-LDP; lane 3, treated with 50 μM of fusion proteinVH-LDP; lane 4, treated with 100 μM of fusion protein VH-LDP; lane 5,treated with 20 μM of 3G11-scFv.

FIG. 9, Preparation of energized fusion protein VH-LDP-AE. ▴, absorbanceat 280 nm; ▪, absorbance at 343 nm.

FIG. 10, Inhibition of bFGF-stimulated angiogenesis by energized fusionprotein VH-LDP-AE in CAM assay. A, PBS control, treated with PBS andbFGF; B, positive control, treated with LDM (0.1 μg/egg) and bFGF; C,treated with VH-LDP-AE (0.5 μg/egg) and bFGF.

FIG. 11. Energized fusion protein VH-LDP-AE inhibited the growth of H22in mice. □, control; ▴, LDM (0.05 mg/kg); Δ, VH-LDP-AE (0.25 mg/kg); x,VH-LDP-AE (0.125 mg/kg); +, mitomycin (1 mg/kg).

FIG. 12: Effects of energized fusion protein VH-LDP-AE plushydroxylcamptothecin on the proliferation of HT-29 cells. ▴,hydroxylcamptothecin; ●, hydroxylcamptothecin+VH-LDP-AE (1 ng/ml);hydroxylcamptothecin+VH-LDP-AE (3 ng/ml); *CDI<0.9; **CDI<0.8;***CDI<0.7.

FIG. 13. Effects of energized fusion protein VH-LDP-AE plus5-fluorouracil on the proliferation of HT-29 cells. ▪, 5-fluorouracil;●, 5-fluorouracil+VH-LDP-AE (1 ng/ml); *CDI<0.9; **CDI<0.8; ***CDI<0.7.

EXAMPLE 1 Cloning of VH Domain Gene and LDM Apoprotein LDP Gene andConstruction of Recombinant Plasmid PET-VH-LDP

Recombinant plasmid pKFv1027 and pIJ1027GRGDS contained the VH gene andLDP gene, respectively, and were both constructed by our laboratory (thepresent laboratory will furnish with the same to the public and providerelated document). Vector pGEM-T is from Promega Company, E. coli DH5αis stored at our laboratory. The express vector is from InvitrogenCompany and stored at our laboratory. PCR primer is synthesized bySangon Company with recognition sites for corresponding restrictionenzymes (Takara Company) introduced therein. The 5′ primer of VH (PH1,SEQ ID NO: 3): 5′GATA CATATG CAGGTGAAGCTGCAGCAGTCT3′;        NdeI         VH The 3′ primer of VH (PH2, SEQ ID NO: 4):5′CATAGGATCCGCCACCGCC TGAGGAGACGGTGACC GTGGT3′       BamHI  spacer        VH The 5′ primer of LDP (PLD1, SEQ ID NO:5): 5′GATA GGATCC GCGCCCGCCTTCTCCGTCAGT3′         BamHI           LDPThe 3′ primer of LDP3 (PLD2, SEQ ID NO: 6): 5′GTTACTCGAG GCCGAAGGTCAGAGCCACGTG3′         XhoI           LDP

The VH gene fragments with GGGGS flexible spacer sequence added inC-terminal were obtained by PCR reaction wherein the template wasrecombinant plasmid pKFv1027 and the primer was PH1 and PH2 for 5′terminal and 3′ terminal, respectively; at the same time, the LDP genefragments were obtained by PCR amplification in which recombinantplasmid pIJ1027GRGDS was the template and the primer was PLD1 and PLD2for 5′ terminal and 3′ terminal, respectively.

PCR procedure as described below: 1.94° C. for 2 minutes; 2.25 cyclesof: 94° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1 minute; and3.72° C. for 10 minutes.

Two PCR products were purified by DNA glass-milk purification kit(BioDev), connected with pGEM-T (Promega), transformed with E. coliDH5α, and then recombinant plasmids are screened. After being confirmedby DNA sequencing (Sangon), the recombinant plasmids were named aspGEM-T-VH and pGEM-T-LDP, respectively. The pGEM-T-VH was digested withBamHI and XhoI, and then the LDP gene fragments were released. Therecombinant plasmid pET-LDP were obtained by subcloning LDP gene intothe vector pET-30a(+). The pGEM-T-VH was digested with NdeI and BamHI,and the released VH gene fragments were subcloned into vector pET-LDP.And the recombinant expression plasmid pET-VH-LDP were obtained andconfirmed by enzyme digestion (FIG. 1) and DNA sequencing. The fulllength gene of fusion protein was 732 bp and coded for 243 amino acids,wherein VH gene was 360 bp and coded for 120 amino acids, gene offlexible spacer was 15 bp and coded for 5 amino acids, LDP gene was 330bp and coded for 110 amino acids, XhoI enzyme digestion site is 6 bp andcoded for 2 amino acids, his-tag gene was 18 bp and coded for 6 aminoacids, and termination codon was 3 bp and coded no amino acid.

In the invention, the XhoI enzyme site was introduced to the 3′-terminalof apoprotein LDP gene of lidamycin, the cluster for 6 successive Hisresidues at multi-cloning site of vector pET-30a(+) would be used well,so there was a His₆-Tag in the 3′-terminal of the fusion protein for itspurification and identification.

EXAMPLE 2 Inducible Expression of the Fusion Protein VH-LDP in E. ColiBL21star™(DE3)

The E. coli strain BL21star™(DE3) in the present invention is fromInvitrogen. The recombinant plasmid pET-VH-LDP was transformed into E.coli BL21star™(DE3) and the recombinant transformant was obtained.Single colony of the transformant was transferred into LB mediumcontaining 30 Hg/ml of kanamycin and cultured overnight at 37° C. Nextday the strains were inoculated by a volume of 1:50 and cultured at 37°C. until the OD₆₀₀ was 0.7. Isopropyl β-D-thiogalactopyranoside (IPTG)was added to a final concentration of 0.05 mM. After 3 hours of growth,the total cell protein sample, medium sample, periplasmic fraction,cytoplasmic solube fraction, cytoplasmic insoluble fraction (inclusionbodies) of the cultured E. coli were prepared in accordance with pETsystem manual (Novagen, 9^(th) edition). And the expression of exogenousprotein was analyzed by 15% SDS-PAGE under reducing conditions. Theresults showed that there was exogenous protein expression in therecombinant E. coli after IPTG induction and the expression yield wasover 30% of total cell protein. The expression protein was insolubleinclusion bodies of E. coli (FIG. 2).

For Western-blot assay, after SDS-PAGE, the proteins were transblottedonto a PVDF membrane under constant current about 0.65 mA/cm² for 1 hourand 50 minutes. The PVDF membrane was incubated with anti-His₆-Tag mAbdiluted (1:2000) in blocking buffer as primary antibody and then withHRP-conjugated goat anti-mouse IgG as secondary antibody, whereupon themembrane was visualized. It was confirmed that the recombinant strainexpressed successfully the fusion protein VH-LDP with a His6-Tag in theC-terminal (FIG. 2).

One transformant strain named CAMS/HLDFP that express fusion proteinVH-LDP was deposited at China General Microbiological Culture CollectionCenter (CGMCC, zhongguancun beiyitiao, Beijing) on Apr. 9, 2004 withaccession number CGMCC No. 1130.

EXAMPLE 3 Purification and Refolding of the Fusion Protein VH-LDP

The fusion protein VH-LDP was purified under denaturing conditions byHis-Bind purification kit (Novagen), and the purification process wasoperated following the user's guide. After being pretreated, theinclusion body sample was loaded to the Ni-NTA column. The column waswashed successively with: (1) 10 vol. of binding buffer (5 mM imidazole,0.5 M NaCl, 20 mM Tris-HCl, 6 M urea, pH 7.9), (2) 6 vol. of washingbuffer (60 mM immidazole, 0.5 M NaCl, 20 mM Tris-HCl, 6 M urea, pH 7.9).The purified protein was finally eluted with 6 vol. of eluting buffer(100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HCl, 6 M urea, pH 7.9) (FIG. 3).The theoretical MW of fusion protein VH-LDP was 25.4 kDa.

The fusion protein VH-LDP after purification was refolded: the purifiedprotein was diluted to a final concentration of 15 μM with elutingbuffer mentioned above, and β-mercaptoethanol was added to a finalconcentration of 10 mM. After being stored at room temperature for 30min, the sample was placed in a dialysis bag and dialyzed against atleast 50 vol. of refolding buffer 1 (50 mM Tris-HCl pH 8.0, 1 mM EDTA,200 mM NaCl, 6 M urea). And the following dialysis was against the samebuffer with step-wise reduction in the urea concentration (3M, 2M, 1M,0.5M, and 0M). On a 1-M stage, 750 μM of glutathione (GSSG) and 400 mMof L-Arginine were added to the dialysis buffer. Then the sample wasdialyzed against 50 vol. of PBS solution (pH 7.4). Each dialysis wasperformed for 12 h and with buffer changed twice. All dialysis wasperformed at 4° C. After dialysis, the sample was centrifuged at 10,000g for 30 min at 4° C. and the supernatant was collected. After beingcondensed, the active fusion protein VH-LDP was obtained and stored at−20° C. for later use.

EXAMPLE 4 Immunoreactivity of Fusion Protein VH-LDP with Type IVCollagenase and Tumor Cells

Immunoreactivity of fusion protein VH-LDP was detected by ELISA. Atfirst, type IV collagenase or tumor cells was coated or fixed to 96-wellELISA plates: well was coated with 100 μl of type IV collagenase (10μg/ml, diluted by PBS) and the plates were stored at 4° C. overnight;Human hepatoma SMMC-7721 cells or human KB cells were seeded at 10⁴/wellin 96-well plates and cultured for 24 h, washed with PBS for 3 times,added 0.05% cold (4° C.) Glutaraldehyde and fixed for 15 min. Then thecoated or fixed plates were washed with PBS 3 times, and blocked with200 μl/well of 1% bovine serum albumin (BSA)/PBS at 4° C. overnight. Theplates were washed 3 times with PBS. Then 200 μl of serialconcentrations of fusion protein VH-LDP were added into each well of theplates and incubated for 2 hours at 37° C. Washing the plates 3 timeswith 0.05% Tween 20/PBS (PBST), adding 50 μl of anti-His₆-Tag monoclonalantibody diluted 1:1500 to each well, and incubating at 37° C. for 1 h.Washing the plates 3 times with PBST, adding 50 μl of HRP conjugatedgoat anti-mouse IgG (at 1:2000 dilution), and incubating at 37° C. for 1hour. Washing the plates with PBST 6 times, adding 100 μl of OPDsubstrate reaction solution to each well, incubating the plates at roomtemperature for 10 min in the dark. Stopping the reaction with 100 μl ofH₂SO₄ (2 mol/L). Then the optical density (OD) of each well was measuredat 490 nm using a microplate reader.

The results demonstrated that the binding activities of fusion proteinVH-LDP to type IV collagenase (FIG. 4), human hepatoma SMMC-7721 cells(FIG. 5), and human oral epidermoid carcinoma KB cells (FIG. 6) were allpositive.

EXAMPLE 5 Immunoreactivity of the Fusion Protein VH-LDP with the SolidTumor of Mouse Hepatoma 22

Immunohistochemical (IHC) staining was performed bystreptavidin-biotin-peroxidase complex (SABC) staining with SABC kit(Boster Company). The sections were blocked by normal goat serum for 20min at room temperature and the excessive liquid was removed. Thesections without washing were added with diluted said VH-LDP fusionprotein as prepared in example 3 and incubated at room temperature; thendiluted anti-His₆-tag antibody and biotinylated goat-anti-mouse IgG wereadded in turn. Finally SABC reagent was added. DAB kit was used as achromogen for visualized reaction at room temperature. The sections werecounter-stained by hematoxylin, dehydrated, cleared, sealed, andobserved with microscope. The results showed that fusion protein VH-LDPwas positively stained in mouse hepatoma sections, and negativelystained in mouse normal liver sections (FIG. 7). This indicated thatVH-LDP has selectivity to tumor tissues.

EXAMPLE 6 Inhibition of Type IV Collagenase Activity by Fusion ProteinVH-LDP

Gelatin zymography protocol was used. Exponentially growing HT-1080cells were seeded at 10⁵/well in 24-well plates and incubated at 37° C.in 5% CO₂. After 24 h, the culture medium was removed and 1 mlserum-free RPMI 1640 medium was added for washing twice. Add 120 μl ofserum-free RPMI 1640 medium and 30 μl of fusion protein VH-LDP asprepared in example 3 to each well, and add 30 μl PBS to control well.Culturing the cells at 37° C. for 24 h. Harvest the culture medium, andcentrifuging at 500 g for 5 min. Then take the supernatant fornon-denaturalization electrophoresis on SDS-PAGE. Wash the gels withdistilled water 3 times. Put the gels into 100 ml of 2.5% TritonX-100,shake on a shaker for 30 min at low speed. Then Wash the gels 2 timeswith distilled water; repeat the washing process with TritonX-100 above.The gels was washed with distilled water 2 times, and incubated at 37°C. for 16-18 h in 100 ml of gelatinase buffer (50 mM Tris-HCl, pH 7.5,200 mM NaCl, 10 mM CaCl₂, 1 μM ZnCl₂). The gels were stained withCoomassie-brilliant blue R-250 and destained with acetic acid:methanol:water (10:45:45). The negative stained bands were observed.

The results showed that fusion protein VH-LDP could inhibitsignificantly the activity of type IV collagenase secreted by cancercells. Compared with the control, the negative stained bands for type IVcollagenase was weakened, and the extent of inhibition was dependent onthe drug concentration (FIG. 8).

EXAMPLE 7 Preparation of Lidamycin (LDM)

The LDM-producing strain (with accession number CGMCC NO. 0135,published in Chinese Patent application 00121527.2) in the frozen-driedtube was resuspended in 0.7 ml salt-free water, transplanted into theGause's NO. 1 slant medium and grown at 28° C. for about 7-10 days. Thenthe aerial mycelium of the strain was transferred to 100/500 ml flaskcontaining the following medium: 1% starch, 0.5% corn syrup, 0.5%peptone, 0.5% glucose, 0.02% MgSO₄, 0.06% KI, 1.5% corn starch, 0.4%CaCO₃, pH 7.0. Shaking fermentation for 48 h at 28° C. then transferredto 1000/5000 ml flask by the volume of 5% and shacked about 18 h at 28°C. in the same medium. After that the producer was transferred to a 200L fermentation tank containing 100 L of medium by the volume of 2% and0.03% defoaming agent was added. The fermentation parameters include:pressure, 0.04; temperature, 28° C.; stirring speed, 400 rpm, air flow1/1, pH 6.5-7.0, fermentation time, 96 hours. 10 L fermented liquid wascentrifuged and the pH of the supernatant was adjusted to 4.0 using HCl.Then 4.5 kg of (NH₄)₂SO₄ was added, and the liquid was stirred for 3 hat 8° C. The precipitated LDM was separated by centrifugation at 8000rpm at 4° C. for 15 min. The pellet was dissolved in 200 ml cold waterand dialyzed. Then the unsolvable sediment was removed bycentrifugation. The supernatant was absorbed by hydroxyl apatite column,eluted by 0.001M PBS (pH6.8) and frozen-dried. Then 1500 mg crudeproduct dissolved in water was loaded to Sephadex G-75 column. Theactive part was frozen-dried. The refined LDM product with highanti-tumor activity was about 145 mg.

EXAMPLE 8 Preparation of Energized Fusion Protein VH-LDP-AE

10 mg of frozen-dried LDM with high activity as obtained in Example 7were added into 5 ml cold methanol and shaken for 5 min, placed at −20°C. for 1 hour. Shake once during the course. Centrifuge at 12,000 r/minat 0° C. for 20 min. The supernatant was rich in chromphore and thesediment contained the apoprotein. The extraction was repeated twice.Chromophore in methanol was vapored and concentrated, stored at −70° C.The chromophores were labile, and the experiments were performed at 4°C. and prevented from illumination. Fusion protein VH-LDP from example 3was dissolved in PBS (0.01 M, pH 7.4), and 5 times ofchromophore-in-methanol by molecular ratio were added to theVH-LDP-containing PBS solution with the volume ratio of 1:50. Aftershaking, the mixture was placed at room temperature for 12 hours. Thenseparation and purification were performed using PD-10 (Sephadex G-25column, Pharmacia) and detected the absorbance at 280 nm and 343 nm.Then the energized fusion protein VH-LDP-AE was collected (FIG. 9). Thetheoretical molecular weight of VH-LDP-AE was 26.2 kDa.

EXAMPLE 9 Cytotoxicity of Energized Fusion Protein on Cancer Cells InVitro

MTT assay was used. After digestion and counting, cells in exponentialgrowth phase were seeded at 3000 cells/well in 96-well plates, andcultured at 37° C. in 5% CO₂ for 24 hours. Drugs of variousconcentrations were added with 3 aliquots, and the cells were culturedfor another 72 h. 50 μl of MTT (2 mg/ml) in serum-free RPMI 1640 wereadded into each well, and the cells were cultured at 37° C. for 4 h.Remove the culture medium gently; add 150 μl DMSO each well; incubate atroom temperature for 15 min on a shaker; measure the absorbance at 560nm on a microplate reader. Survival ratio and IC₅₀ values werecalculated according to the following equation: Survivalratio=(A_(test)−A_(blank))/(A_(control)−A_(blank))×100%. The resultsindicated that energized fusion protein VH-LDP-AE displayed extremelypotent cytotoxicity to cancer cells. As table 1 demonstrated, the IC₅₀values to HT-1080 cells, KB cells, and PG cells were all lower than1×10¹¹ M (Table 1). TABLE 1 The cytotoxicity of VH-LDP-AE to cancercells IC₅₀(M) Groups HT-1080 KB PG LDM 2.21 × 10⁻¹² 1.08 × 10⁻¹² 6.30 ×10⁻¹² VH-LDP-AE 7.65 × 10⁻¹³ 4.35 × 10⁻¹⁴ 1.49 × 10⁻¹³

EXAMPLE 10 Anti-Angiogenic Activity of the Energized Fusion ProteinVH-LDP-AE

Anti-angiogenic activity of VH-LDP-AE was examined in chickembryoallantoic membrane assay (CAM). The surface of 7-day-oldpost-fertilization chick eggs (White Leghorn) in a 60% humidifiedincubator at 37° C. was sterilized and the CAM was 2) exposed by cuttinga window (2 cm²) on the egg shell using the false air-sac technique.After 24 h, 10 μl of bFGF was dipply added to agarose disks with theenergized fusion protein VH-LDP-AE at various concentrations which wasprepared as mentioned in Example 7, and then the disks were placed ontop of the CAM. After the windows were sealed with transparent tape, theeggs were incubated for further 72 h. The results shown in FIG. 10indicated that VH-LDP-AE significantly suppress angiogenesis stimulatedby bFGF.

EXAMPLE 11 The Therapeutic Effects of Energized Fusion Protein VH-LDP-AEon the Growth of Transplanted Hepatoma 22 in Mice

Kunming mice, weighing between 18-22 g, were randomly separated intodifferent groups of 10 mice each. On day 0, hepatoma 22 cells (1.5×10⁶cells/0.2 ml/mouse) diluted with saline were transplanted subcutaneouslyinto the right axilla of mice. On day 1 (after 24 h), the therapy wasstarted and VH-LDP-AE at different doses, LDM at 0.05 mg/kg, mitomycin(MMC) at 1 mg/kg were administered respectively to the tumor bearingmice (0.2 ml/mouse) by injection into the tail vein. Mice of controlgroup were injected with saline. Diameters of the tumors were measuredevery 3 days during the experiment. Weights of the mice were recorded.The tumor volumes were calculated with the following formula: V=0.5ab²,where a and b is the long and the perpendicular short diameters of thetumor, respectively. The curves of tumor growth were plotted and theinhibition rates were calculated.

The results showed that both 0.25 mg/kg and 0.125 mg/kg of energizedfusion protein VH-LDP-AE inhibited or retarded the growth of hepatoma 22in mice, and the inhibition efficacy was more potent than the freelidamycin at the maximal tolerated dose of 0.05 mg/kg. This indicatedthat VH-LDP-AE can enhance the therapeutic effects of lidamycin (FIG.11). The results on day 14 were shown on Table 2: TABLE 2 Inhibition ofthe growth of transplantable hepatoma 22 by VH-LDP-AE in mice Doses Micenumber Body weight (g) Tumor volume (cm³) Inhibition (mg/kg) begin/endbegin/end x ± s rate (%) Control 10/10 18.89/33.20 4.60 ± 1.48 LDM 0.0510/10 19.77/26.28 0.94 ± 0.53 79.6^(ΔΔ) VH-LDP-AE 0.25 10/10 19.27/23.500.19 ± 0.21 95.9**^(ΔΔ) 0.125 10/10 19.01/27.59 0.55 ± 0.24 88.1*^(ΔΔ)MMC 1 10/10 18.82/29.29 2.23 ± 2.15 51.5^(Δ)P < 0.05 vs. LDM, indicated by *;P < 0.01 vs. LDM, indicated by **P < 0.05 vs. control, indicated by Δ;P < 0.01 vs. control, indicated by ΔΔ

The inhibition rates of tumor growth of VH-LDP-AE was 95.9% and 88.1%,at doses of 0.25 mg/kg and 0.125 mg/kg, respectively, the effects weremore potent than that of LDM (79.6%), and mitomycin, used aschemotherapeutic drug in clinics, showed the inhibition rate of 51.5%(Table 2). No body weight loss and other severe side-effects were foundduring the experiments. This indicates that mice well tolerated thedoses.

EXAMPLE 12 The Inhibitory Effects of VH-LDP-AE in Combination withVarious Anticancer Drugs on Cancer Cell Proliferation

MTT assay was used. After trypsin-EDTA digestion, the human coloncarcinoma HT-29 cells in exponential growth were seeded at 4000cells/well in 96-well plates, and cultured for 24 hours. Then add 20 μlof different concentrations of hydroxylcamptothecin or 5-fluorouracil; 8hours later, add different concentrations of VH-LDP-AE. Continue toculture the cells at 37° C. in 5% CO₂ for 72 h. Add 50 μl of MTT atconcentration of 2 mg/ml, culture the cells at 37° C. for 4 hours. Thenremove the supernatant; add 150 μl DMSO each well; ten minutes later,measure the absorbance at 560 nm on a microplate reader. In cancerpharmacology, interactions of drug combination were evaluated bycoefficient of drug interaction (CDI). There existed synergistic effectswith CDI<1, and there existed very significant ones with CDI<0.7. Forthe combination of hydroxylcamptothecin (1 μM) and energized fusionprotein VH-LDP-AE (3 ng/ml), CDI value was less than 0.7, and whichindicated that they can inhibited synergistically the proliferation ofHT-29 cells (FIG. 12). For 5-fluorouracil plus VH-LDP-AE, the CDI of 10μM of 5-fluorouracil and 3 ng/ml of VH-LDP-AE was less than 0.7, andwhich showed that there existed a very obvious synergistic effectsbetween them (FIG. 13). It was usual that monoclonal antibodies werecombined with anti-cancer drugs. The present results showed that thereexisted significantly synergistic effects between energized fusionprotein VH-LDP-AE and 5-fluorouracil or hydroxylcamptothecin.

1. A single domain antibody energized fusion protein VH-LDP-AE consisting of a fusion protein VH-LDP that contains the heavy chain variable domain VH of monoclonal antibody 3G11 against type IV collagenase, the flexible spacer GGGGS, the apoprotein of lidamycin (LDP), and a His₆-tag tail; and an active enediyne chromophore (AE) that derives from lidamycin.
 2. The single domain antibody energized fusion protein VH-LDP-AE of claim 1, wherein the gene sequence coding for said fusion protein VH-LDP is set forth in SEQ ID NO: 1, the amino acid sequence thereof is set forth in SEQ ID No:
 2. 3. The single domain antibody energized fusion protein VH-LDP-AE of claim 1, wherein the molecular ratio of said fusion protein and active chromophore AE of lidamycin is 1:1.
 4. A method for producing single domain antibody energized protein VH-LDP-AE of claim 1, comprising: 1) Preparing the fusion protein VH-LDP containing single domain antibody against type IV collagenase; 2) Preparing the active chromophore AE of lidamycin by the method of cold methanol extraction; 3) Mixing the resultant active chromophore AE of lidamycin extracted by cold methanol in solution of methanol and VH-LDP in 0.01 M PBS (pH7.4) by a molecular ratio of 1:5 and a volume ratio of 50:1, reacting in dark at room temperature for 12 hours, and the energized fusion protein VH-LDP-AE is obtained.
 5. Use of single domain antibody energized fusion protein VH-LDP-AE of claim 1 in manufacture of anti-angiogenic and anti-cancer targeting drug.
 6. The use of claim 5, wherein said tumor is selected from the group consisting of solid tumors including digestive tract cancers, such as hepato-carcinoma, colon carcinoma, rectum carcinoma, esophageal carcinoma, gastric carcinoma; breast carcinoma; ovarian carcinoma; lung carcinoma; and renal carcinoma.
 7. A pharmaceutical composition comprising therapeutically effective amount of single domain antibody energized fusion protein VH-LDP-AE of claim 1, and optionally, pharmaceutical acceptable carrier and/or excipient.
 8. A method for treating a tumors in a human comprising the step of administering a therapeutically effective amount of single domain antibody energized fusion protein of claim 1 to a patient with the tumor.
 9. A method for treating a tumors in a human comprising the step of administering a therapeutically effective amount of said pharmaceutical composition of claim 7 to a patient with the tumor. 